Over the past 50 years, pharmaceuticals have been developed directly from microbial sources and have had an important influence on health care throughout the world. For example, the penicillin antibiotics have had an enormous impact on the control of bacterial infections in humans and animals. In addition to antibacterial activity, other classes of microbial agents have been discovered and developed that have, for example, antifungal, anticancer, and immunosuppressive activities.
Over the past two decades, genetic engineering has become an important tool for modifying biological materials to enhance their natural capabilities. For example, in the pharmaceutical area, a number of human proteins (hormones, immunomodulators, enzymes) have been developed by cloning the gene encoding the protein, isolating the protein product and using it directly as a therapeutic agent.
Production of natural metabolites from microbial sources is generally enhanced by strain improvement and process optimization. Classical strain improvement is primarily based on random mutagenesis and subsequent selection for higher producers. Recent development of recombinant DNA technology provides the potential for genetic engineering of microorganisms to enhance the production of metabolites, a more rational alternative to the classical strain improvement techniques.
The following four publications illustrate strain improvement with the use of recombinant DNA technology: 1) Paul L. Skatrud et al., in BIO/TECHNOLOGY, 7, May 1989, pp 477-485, discloses a method of improving production of cephalosporin C in a late step modification by inserting a second copy of a gene encoding a bifunctional protein, expandase/hydroxylase. No disclosure is made to enhance precursor flux. 2) U.S. Pat. No. 5,108,918 relates to a method enhancing production of a secondary metabolite in a bacterial or fungal host by random cloning of a plasmid containing genes for secondary metabolite production. However, this method is non-targeted and non-rational and does not disclose enhancement of precursor flux. 3) A recombinant strain of Penicillium chrysogenum was constructed containing extra copies of a gene encoding isopenicillin N synthetase and enhanced enzyme activity showed no improvement in penicillin production, which suggests that this enzyme is not rate-controlling, disclosed by Paul L. Skatrud et al., Poster presentation 1987 Annual Meeting of Society of Industrial Microbiology, Baltimore, Aug. 1987, abstract published in SIM News (1987) 33:77.4). The art has taught that the amount of starch in plant tissue can be enhanced by increasing the expression of a rate controlling enzyme by use of a regulatory variant of the enzyme. There is no disclosure of inserting a second copy of a cloned gene or of enhancing precursor flux.
Studies have shown significant effects of primary (i.e., precursor) metabolites on the production levels of .beta.-lactam antibiotics. For example, addition of the amino acid, lysine, in the fermentation culture of Penicillium chrysogenum depressed the level of antibiotics, which can be attributed to the feedback inhibition of an enzyme, i.e., homocitrate synthetase, by lysine as disclosed by J. M. Luengo, et al., J. Bacteriol., 144:869-76 (1980), and P. S. Masurekar, et al., Can. J. Microbiol., 18:1045-1048 (1972). In contrast, addition of the amino acids, lysine and DL-meso-diaminopimelic acid (DAP), in Streptomyces clavuligerus stimulated production of the antibiotic cephamycin C as disclosed by S. Mendelovitz, et al., Antimicrob. Agents Chemother., 21:74-84 (1982), possibly by providing a larger precursor pool for biosynthesis of .alpha.-aminoadipic acid (.alpha.-AAA), or as a result of activation of aspartokinase, the first enzyme involved in lysine biosynthesis via the aspartate pathway.
These publications relate to effect of precursor pools on the cephamycin biosynthesis but no disclosure is made of inserting a second copy of a cloned gene.